Fig. 8: Subunit composition of the FRQ-FRH complex.

A FLAG-IPs from HEK293T lysates expressing the proteins schematically indicated in the top panels. Left panels: Control IP from cells expressing mK2-FRH and mK2-FRQ but no FLAG-FRH. Middle panels: Control IP from cells expressing mK2-FRH and FLAG-FRH but no mK2-FRQ. Right panels: FLAG IP from cells expressing mK2-FRH, mK2-FRQ and FLAG-FRH. *: In a substantial fraction of mK2-FRH the mK2-moiety was clipped off, giving rise to an untagged FRH moiety that comigrates with FLAG-FRH. 25 µg total lysate (T) and supernatant (S) and aliquots of the immunoprecipitates corresponding 100 µg (4 × IP) were analyzed by Western blot with antibodies against FRH (upper panel), FRQ (middle panel) and FLAG (lower panel) (n = 3). FLAG visualization was performed on a different gel using the same samples. B GFP pulldown from Neurospora lysates expressing FRH-GFP in addition to endogenous FRH. frh-gfp #2 and #12 represent two independent strains. T, 400 µg total lysate; S, aliquots of supernatant; IP, aliquots of immunoprecipitates corresponding to 1×, 4×, and 8× of the total lysate lane, respectively. Representative results from three independent experiments with reproducible results are shown. C GFP pulldown from frq^WT and from frq^WT, frh-gfp and frq¹⁰, frh-gfp strains. Representative results from three independent experiments with reproducible results are shown. D Model of CK1a-dependent subunit remodeling in the FRQ-FRH complex. Newly synthesized, unphosphorylated FRQ accumulates with FRH in the nucleus as a heterotetramer with an FRQ₂FRH₂ stoichiometry. The nuclear accumulation of FRQ is facilitated by its three nuclear localization signals (NLSs) and is further supported by a tight association with FRH, which provides a nuclear import signal and additionally mask FRQ’s nuclear export signal (NES). The two bound FRH molecules block the WCC binding sites on FRQ, rendering the FRQ₂FRH₂ complex inactive. Consequently, the WCC remains active, promoting frq transcription and supporting the accumulation of high levels of inactive FRQ₂FRH₂. Phosphorylation of FRQ inactivates its NLSs and, after a considerable delay, triggers the dissociation of FRH. Due to the slow and partially stochastic nature of FRQ phosphorylation, the release of the two FRH molecules from a FRQ dimer is not synchronous, leading to the transient formation of a heterotrimeric FRQ₂FRH₁ species. The FRQ₂FRH₁ complex is the active species because it exposes a WCC binding site, allowing CK1a to phosphorylate and inactivate transiently interacting WCCs, thereby shutting down frq transcription. The FRQ₂FRH₁ complex can be exported via the exposed NES in one FRQ subunit but will be reimported via the FRH bound to the second FRQ subunit. Further phosphorylation of FRQ by CK1a eventually triggers the dissociation of the second FRH molecule, resulting in an FRQ₂-only species. At this stage, FRQ’s NLSs are fully inactivated by phosphorylation, prompting relocalization of FRQ₂ to the cytosol, where it can no longer support phosphorylation of nuclear WCC and is eventually degraded, thus closing the negative feedback loop. Subsequent dephosphorylation of WCC initiates a new circadian cycle. Created with BioRender.com.